US5824529A - Method for cloning and producing the PshAI restriction endonuclease - Google Patents

Method for cloning and producing the PshAI restriction endonuclease Download PDF

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US5824529A
US5824529A US08/611,510 US61151096A US5824529A US 5824529 A US5824529 A US 5824529A US 61151096 A US61151096 A US 61151096A US 5824529 A US5824529 A US 5824529A
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pshai
dna
methylase
endonuclease
gene
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Zhiyuh Chang
Richard D. Morgan
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New England Biolabs Inc
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  • the present invention relates to recombinant DNA which encodes the PshAI restriction endonuclease and modification methylase, and the production of PshAI restriction endonuclease from the recombinant DNA.
  • Type II restriction endonucleases are a class of enzymes that occur naturally in bacteria. When they are purified away from other bacterial components, restriction endonucleases can be used in the laboratory to cleave DNA molecules into precise fragments for molecular cloning and gene characterization.
  • Restriction endonucleases act by recognizing and binding to particular sequences of nucleotides (the ⁇ recognition sequence ⁇ ) along the DNA molecule. Once bound, they cleave the molecule within, or to one side of, the recognition sequence. Different restriction endonucleases have affinity for different recognition sequences. Over one hundred and eighty restriction endonucleases with unique specificities have been identified among the many hundreds of bacterial species that have been examined to date.
  • the endonucleases typically are named according to the bacteria from which they are derived.
  • the species Deinococcus radiophilus for example, synthesizes three different restriction endonucleases, named DraI, DraII and DraIII.
  • These enzymes recognize and cleave the sequences TTTAAA (SEQ ID NO:1), PuGGNCCPy (SEQ ID NO:2) and CACNNNGTG (SEQ ID NO:3), respectively.
  • Escherichia coli RY13 synthesizes only one enzyme, EcoRI, which recognizes the sequence GMTTC (SEQ ID NO:4).
  • restriction endonucleases play a protective role in the welfare of the bacterial cell. They enable bacteria to resist infection by foreign DNA molecules like viruses and plasmids that would otherwise destroy or parasitize them. They impart resistance by cleaving invading foreign DNA molecule each time that the recognition sequence occurs. The cleavage that takes place disables many of the infecting genes and renders the DNA susceptible to further degradation by non-specific nucleases.
  • a second component of bacterial protective systems are the modification methylases. These enzymes are complementary to restriction endonucleases and they provide the means by which bacteria are able to protect their own DNA and distinguish it from foreign, infecting DNA. Modification methylases recognize and bind to the same recognition sequence as the corresponding restriction endonuclease, but instead of cleaving the DNA, they chemically modify one or other of the nucleotides within the sequence by the addition of a methyl group. Following methylation, the recognition sequence is no longer cleaved by the restriction endonuclease. The DNA of a bacterial cell is always fully modified by virtue of the activity of its modification methylase. It is therefore completely insensitive to the presence of the endogenous restriction endonuclease. It is only unmodified, and therefore identifiably foreign DNA, that is sensitive to restriction endonuclease recognition and cleavage.
  • the key to isolating clones of restriction endonuclease genes is to develop a simple and reliable method to identify such clones within complex ⁇ libraries ⁇ , i.e. populations of clones derived by ⁇ shotgun ⁇ procedures, when they occur at frequencies as low as 10 -3 to 10 -4 .
  • the method should be selective, such that the unwanted majority of clones are destroyed while the desirable rare clones survive.
  • Type II restriction-modification systems are being cloned with increasing frequency.
  • the first cloned systems used bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcoRII: Kosykh et al., Molec. Gen. Genet 178: 717-719, (1980); HhaII: Mann et al., Gene 3: 97-112, (1978); PstI: Walder et al., Proc. Nat. Acad. Sci. 78 1503-1507, (1981), the disclosures of which are hereby incorporated by reference herein).
  • a third approach which is being used to clone a growing number of systems involves selection for an active methylase gene (refer to our EPO No.: 193,413 published, Sep. 3, 1986 and BsuRl: Kiss et al., Nucl. Acid. Res. 13: 6403-6421, (1985), the disclosures of which are hereby incorporated by reference herein). Since restriction and modification genes are often closely linked, both genes can often be cloned simultaneously.
  • coli strain ER1992 contains a dinD1-Lac Z fusion but is lacking the methylation dependent restriction systems McrA, McrBC and Mrr.
  • McrA methylation dependent restriction systems
  • McrBC methylation dependent restriction systems
  • Mrr methylation dependent restriction systems
  • the endonuclease gene can be detected in the abscence of it's cognate methylase when the endonuclease damages the host cell DNA, inducing the SOS response.
  • the SOS-induced cells form deep blue colonies on LB agar plates supplemented with X-gal. (Fomenka et al. Nucleic Acids Res. 22:2399-2403 (1994), the disclosure of which is hereby incorporated by reference herein).
  • methylase (and/or endonuclease) selection method fails to yield a methylase (and/or endonuclease) clone due to various obstacles. See, e.g., Lunnen, et al., Gene, 74(1):25-32 (1988), the disclosure of which is hereby incorporated by reference herein.
  • One potential obstacle to cloning restriction-modification genes lies in trying to introduce the endonuclease gene into a host not already protected by modification. If the methylase gene and endonuclease gene are introduced together as a single clone, the methylase must protectively modify the host DNA before the endonuclease has the opportunity to cleave it. On occasion, therefore, it might only be possible to clone the genes sequentially, methylase first then endonuclease.
  • a third potential difficulty is that some restriction endonuclease and methylase genes may not express in E. coli due to differences in the transcription machinery of the source organism and E. coli, such as differences in promotor and ribosome binding sites.
  • the methylase selection technique requires that the methylase express well enough in E. coli to fully protect at least some of the plasmids carrying the gene.
  • the present invention relates to recombinant DNA encoding the genes for the PshAI restriction endonuclease and modification methylase obtainable from Plesiomonas shigelloides as well as related methods for the production of these enzymes from the recombinant DNA.
  • This invention also relates to a transformed host which expresses the restriction endonuclease PshAI, an enzyme which recognizes the DNA sequence 5' GACNNNNGTC 3' (SEQ ID NO:5) and cleaves at the middle of the recognition sequence to produce blunt ends.
  • PshAI restriction endonuclease produced according to the present invention is substantially pure and free of the contaminants normally found in restriction endonuclease preparations made by conventional techniques.
  • the PshAI methylase gene was obtained generally in accordance with the technique referred to as methylase selection (U.S. Pat. No. 5,200,333, the disclosure of which is hereby incorporated by reference herein). However none of the clones obtained by methylase selection expressed detectable PshAI restriction endonuclease activity.
  • the N-terminal amino acid sequence of the PshAI endonuclease protein was needed.
  • a protein purification method was developed to purify the PshAI endonuclease to near homogeneity from Plesiomonas shigelloides.
  • the purified PshAI endonuclease was used to determine the N-terminal amino acid sequence of PshAI endonuclease. This amino acid sequence was compared with amino acid translation of the DNA sequence of the methylase clones obtained from the methylase selection technique.
  • DNA contiguous to the PshAI methylase clones was amplified by inverse PCR techniques, cloned in pieces so as to not contain an intact endonuclease gene and sequenced.
  • An open reading frame in which the deduced amino acid sequence matched the N-terminal amino acid sequence of PshAI endonuclease was located 5' of the methlase gene and separated from the methylase by a gene having homology to the EcoRV control gene. (Bougueleret, et al., supra).
  • the PshAI endonuclease gene was then cloned into an appropriate expression vector and introduced into a host which was pre-modified with the PshAI methylase carried on a separate, compatible vector.
  • the preferred method for cloning the PshAI restriction-modification system consists of creating a vector containing multiple PshAI sites and cloning the PshAI methylase by methylase selection.
  • the DNA sequence of PshAI methylase positive clones is determined.
  • the PshAI endonuclease is purified to near homogeneity, and the amino acid sequence at the N-terminus of the protein is determined.
  • DNA 5' to the methylase clones is amplified by inverse PCR techniques, cloned and sequenced.
  • the PshAI endonuclease gene is identified based on the DNA sequence and amino acid sequence data.
  • the PshAI endonuclease can then be expressed by amplifying the complete gene from Plesiomonas shigelloides DNA and cloning it into an expression vector such as pRRS.
  • This construct is introduced into a host which is premodified at PshAI sites by virtue of a PshAI methylase gene carried on a separate compatible plasmid.
  • PshAI endonuclease is produced by growing the host containing the PshAI endonuclease and methylase genes, inducing with the appropriate expression conditions, harvesting purifells and purifying the PshAI endonuclease.
  • FIG. 1 illustrates the preferred method for cloning and producing the PshAI restriction endonuclease.
  • FIG. 2 is a restriction map of the Plesiomonas shigelloides DNA contained in the methylase clone pPshAIM19.
  • This clone pPshAIM19 is obtained by methylase selection and contains a full length PshAI methylase. The location and orientation of the PshAI methylase and also ORF1 are shown.
  • FIG. 3 is shows the locations and orientations of the PshAI methylase gene, the control gene and the endonuclease gene.
  • FIG. 4 lists the amino acid sequences of PshAI (SEQ ID NO:6) and EcoRV (SEQ ID NO:7) control proteins.
  • FIG. 5 is a photograph of an agarose gel demonstrating PshAI restriction endonuclease activity in cell extracts of E. coli ER2426 carrying the PshAI endonuclease on the pRRS derived plasmid pPshAIR1 and the PshAI methylase on the pSX34laz ⁇ derived plasmid pSX34PshAIM1.
  • 1.5 gram of cell was suspended in 20 ml of sonication buffer (20 mM Tris-HCl, 1 mM dithiothreitol, 0.1 mM EDTA, pH 7.5) and broken by sonication, and clarified by centrifugation.
  • the present invention relates to recombinant DNA which encodes the PshAI restriction endonuclease and methylase, as well as to the enzymes produced from such a recombinant DNA.
  • the endonuclease protein was purified to near homogeneity and used to determine amino acid sequence at the amino-terminal end of the protein.
  • the amino acid sequence obtained was compared with the DNA sequence adjacent to the methylase gene.
  • the N-terminal amino acid sequence of the ORF1 did not match with the PshAI protein sequencing data, nor was any match to the PshAI protein sequence data found in the DNA sequence of clone pPshAIM19.
  • the 1 kb EcoRI to DraI fragment was cloned into vector pUC19 and sequenced.
  • oligonucleotide primers complementary to regions of known DNA sequences flanking this fragment were used to amplify this DNA by PCR, and the amplified product was cloned into pUC19 and sequenced.
  • the amino acid sequence obtained from protein sequencing of PshAI endonuclease was compared with the six frame amino acid translation of the DNA sequence 5' to the methylase gene.
  • An open reading frame of 888 bp, ORF2 was identified in which the first 28 amino acid residues matched the amino-terminal sequence of the PshAI endonuclease.
  • the PshAI endonuclease (ORF2) was separated from the PshAI methylase by an intervening open reading frame of 237 bp (ORF3) with significant homology to the EcoRV control (C) gene (FIG. 4).
  • ORF3 is believed to be a PshAI control gene.
  • the PstI site is located on the polylinker region of the pUC19PshAI vector) containing the entire methylase gene was cloned into the vector pSX34lacz ⁇ (New England Biolabs, Inc.; Beverly, Mass.) which has a pSC101 type origin of replication. Clones expressing PshAI methylase were selected by the methylase selection method.
  • the extracts were assayed for PshAI endonuclease activity.
  • One PshAI expressing host, pPshAIRl was propagated and used to produce PshAI restriction endonuclease.
  • the PshAI endonuclease was purified by a protein purification scheme described herein below.
  • FIG. 1 The method described herein by which the PshAI restriction endonuclease and methylase genes are preferably cloned and expressed is illustrated in FIG. 1 and includes the following steps:
  • the PshAI restriction endonuclease is partially purified from Plesiomonas shigelloides cells by a combination of protein purification techniques developed at New England Biolabs, Inc. (Beverly, Mass.) (see Example 1, step 2).
  • Random libraries of Plesiomonas shigelloides DNA are constructed.
  • a vector containing three PshAI sites, pUC19PshAI is formed by introducing a DNA oligomer containing a PshAI site into the SmaI, SspI and DraI (1563 or 1582) sites of pUC19.
  • Plesiomonas shigelloides DNA is partially digested with HinP1I, Sau3AI and NlaIII respectively to produce fragments of an average size ranging from 2 kb-10 kb. These fragments are ligated with the vector pUC19PshAI.
  • the ligated DNA is transformed into E. coli, the transformants are pooled and the populations of plasmids are purified to form libraries.
  • the methylation selection method is used to select for PshAI methylase clones.
  • Each of the HinP1I, Sau3AI and NIallI libraries are digested with PshAI endonuclease.
  • the PshAI restricted plasmids are transformed back into E. coli to recover undigested clones.
  • a number of individual transformants of plasmids surviving PshAI digestion are grown and mini-preparations are made of their plasmids.
  • the plasmids are analyzed for resistance to PshAI endonuclease digestion. Two clones which are protected from PshAI cleavage and contain similarly sized HinP1I fragments are found.
  • clones, pPshAIM19 and pPshAIM26 from the HinP1I library contain inserts 3 kb and 4.5 kb respectively.
  • the methylase positive clones are assayed for PshAI restriction endonuclease activity, but no activity can be detected.
  • the PshAI restriction endonuclease protein is purified to near homogeneity from Plesiomonas shigelloides by a combination of protein purification techniques developed at New England Biolabs (see Example 1, step 6).
  • the endonuclease so purified is nearly homogeneous on SDS polyacrylamide gel electrophoresis and has an apparent molecular weight of approximately 35 kilodaltons.
  • the amino terminal amino acid sequence of the endonuclease is obtained using an Applied BioSystems Division, Perkin-Elmer Corporation (Foster City, Calif.) 470A Protein Sequencer (Brooks, et al., Nucleic Acids Research, 17:979-997 (1989), the disclosure of which is hereby incorporated by reference herein), and used to identify PshAI endonuclease gene in subsequent studies.
  • the amino acid sequence of PshAI did not match with amino acid sequence deduced from the DNA sequence adjacent to the methylase gene on the methylase clones pPshAIM19.
  • Plesiomonas shigelloides DNA is digested with various restriction endonucleases. The digests are electrophoresed on an agarose gel and then transferred to immobilon-S transfer membrane (Millipore Corporation; Bedford, Mass.). Fragments containing DNA extending 5' to the methylase gene a30 hybridization, n hybridization, using a portion of pPshAIM19 as probe. A 2.9 kb AvrII fragment which should contain 2.1 kb of sequence 5' to pPshAIM19 is identified.
  • the circularized 2.9 kb fragment containing DNA corresponding to pPshAIM19 is amplified using two synthetic primers which anneal to the known sequence region and are oriented with their 3' ends toward the unknown region.
  • restriction gene can be overexpressed.
  • the DNA sequence and detailed mapping information help determine the best approach for overexpression of the restriction endonuclease gene.
  • One approach for overexpression comprises designing primers that hybridize directly at the N-terminus of the restriction endonuclease gene and somewhere downstream (3') of the gene in order to use the polymerase-chain reaction to amplify the entire endonuclease gene.
  • the resulting DNA fragment can be inserted into an expression vector such as pAII17 directly downstream of an inducible promoter (T7).
  • the host is generally pre-protected from restriction endonuclease digestion. In the present invention this is accomplished by cloning the PshAI methylase on a separate plasmid.
  • the plasmid used must be compatible with the expression vector.
  • the methylase also must be produced at a level which will protect the host's genome from digestion by the overexpressed restriction endonuclease gene.
  • the DNA sequence of the gene can be altered by site-directed mutagenesis or by resynthesizing the gene itself to use codons that are more efficiently utilized in E. coli (Ikemura, J. Mol. Biol. 151:389-409 (1981), the disclosure of which is hereby incorporated by reference herein).
  • the PshAI methylase clone, pPshAIM19 is digested with PstI and Xbal to generate a 1660 bp fragment containing the entire methylase gene. This fragment is cloned into vector pSX34Iacz ⁇ (New England Biolabs, Inc.; Beverly, Mass.). A population of colonies is subjected to one round of methylase selection and clones expressing the PshAI methylase are identified by introducing the vector pUC19PshAI (which contains three PshAI sites) into E. coli cells containing pSX34lacz ⁇ methylase constructs, performing minipreps and digesting with PshAI.
  • Competent cells are made from a single clone, designated pSX34PshAIM1, for subsequent PshAI endonuclease expression.
  • DNA primers are designed and synthesized to amplify the entire PshAI endonuclease gene.
  • the forward primer has the following elements: a PstI cloning site, stop codon in frame with the lacZ gene, E. coli consensus strong ribosome binding site, 7 nucleotide spacer sequence between the ribosome binding site and the ATG start codon of the PshAI endonuclease, a change of codo a usage in amino acid number 2 to an E. coli preferred codon and 19 nucleotides matching the PshAI endonuclease DNA sequence for hybridization.
  • the 3' primer is designed to hybridize Plesiomonas shigelloides DNA approximately 200 bp beyond the 3' end of the endonuclease gene. BamHI and EcoRI sites were introduced in the reverse primer to facilitate cloning.
  • the endonuclease gene is amplified from the genomic DNA using these primers.
  • the amplified DNA is cleaved by PstI and BamHI and ligated into the expression vector pRRS, which has been previously cleaved by the same enzymes and gel purified.
  • the ligation reaction is transformed into E. coli ER2426 competent cells containing pSX34PshAIM1. Vectors containing inserts of the desired size are identified by miniprep procedures.
  • PshAI endonuclease activity is assayed for PshAI endonuclease activity.
  • PshAI expressing host designated pPshAIR1
  • pPshAIR1 is propagated and used to produce PshAI restriction endonuclease.
  • the PshAI endonuclease may be produced from host cells carrying the overexpressed PshAI restriction endonuclease gene and PshAI methylase gene by propagation in a fermenter in a rich medium with the appropriate antibiotic selection and induction. The cells are thereafter harvested by centrifugation and disrupted by sonication to produce a crude cell extract containing PshAI restriction endonuclease activity.
  • the crude cell extract containing the PshAI endonuclease is purified by a combination of protein purification techniques, such as affinity-chromatography or ion-exchange chromatography.
  • DNA purification A single colony of Plesiomonas shigelloides from an agar plate was innoculated into 1 L LB broth, grown overnight with shaking at 37° C. and the cells were pelleted by centrifugation. 3 g of cell paste was resuspended by gentle shaking in 20 ml of 25% sucrose, 0.05 M Tris-HCl, 1 mM EDTA, pH 8.0. 5 ml of 0.5M EDTA, pH 8.0 and 6 ml of freshly prepared 10 mg/ml lysozyme in 0.25 M Tris-HCl pH 8.0 was added and the solution was incubated at 4° C. for 2 hours.
  • Triton-X100 40 ⁇ l of 25% Triton-X100 was added to a final concentration of 0.1%.
  • the cell suspension was frozen at -70° C. then thawed on ice.
  • the extract was centrifuged at 15,000 rpm for 15 minutes at 40° C. and the supernatant was passed through a 0.22 micron filter.
  • the supernatant was then loaded onto a 1 ml HiTrapTM Heparin column (Pharmacia; Piscataway, N.J.) equilibrated with buffer A containing 50 mM NaC.
  • the column was washed with 10 ml of buffer A containing 50 mM NaCl, followed by a 40 ml linear gradient from 0.05M NaCl to 1M NaCl in buffer A.
  • the PshAI restriction enzyme activity eluted between 0.2 and 0.25M NaCl. Fractions containing PshAI were mixed with an equal volume of glycerol and stored at -20° C. The HiTrapTM Q fractions were assayed on ⁇ DNA and found to contain approximately 6000 units of PshAI.
  • PshA I linker 5'-pGACGGCCGTC-3' (SEQ ID NO:8)
  • the cleaved pUC19 DNAs was dephophorylated with Calf Intestinal Alkaline Phosphatase (CIP) according to the manufacturer's instruction.
  • CIP Calf Intestinal Alkaline Phosphatase
  • 1 ⁇ l of 1 ⁇ M annealed linker was ligated with each digested and dephophorylated vector pUC19 (50 ng) in a 20 ⁇ l reaction volume using 400 U (New England Biolabs, Inc.; Beverly, Mass.) of T4 DNA ligase for 2 hours at 16° C.
  • the ligation mixture was transformed into E. coli strain ER 2420 and plated on L-broth plates containing 100 ⁇ g/ml ampicillin for individual colonies. Clones of the desired construct were identified by performing minipreps, digesting the purified DNA with PshAI and analyzing it by agarose gel electrophoresis.
  • the miniprep DNA of the desired clones was digested with EagI and gel purified. 20 ng of the digested DNA was used to self-ligate in a 20 ⁇ l reaction volume using 400 U T4 DNA ligase at 16° C. overnight. The ligation mixture was transformed into E. coli ER2420 and plated on L-broth plates containing ampicillin (100 ⁇ g/ml). Plamids were isolated from individual colonies, using the miniprep procedure as described. Clones of the desired construct were identified by digesting the plasmid with PshAI and analyzing by agarose gel electrophoresis. Plasmid from a clone of each construct, designated pSmaI, pDraI and pSspI respectively, was used in the subsequent steps.
  • 1.5 ⁇ g of the gel purified NlaIII, Sau3AI, HinP1I partially digested DNA was ligated to 0.5 ⁇ g vector Puc19PshAI which had been previously cleaved by SphI, BamHI and AccI respectively and dephosphorylated with calf intestinal alkaline phosphatase in 200 ⁇ l of 1 ⁇ T4 DNA ligase buffer containing 1000 units of T4 DNA ligase (New England Biolabs, Inc.; Beverly, Mass.) for 16 hours at 17° C. 30 ⁇ l the of ligation reactions was transformed into ER2426 and plated on L-broth plates containing 100 ⁇ g/ml ampicillin.
  • 352 ⁇ l of the cleared supernatant was transfered into a fresh tube and mixed with 88 ⁇ l of 4M NaCl and 440 ⁇ l of 13% PEG 8000 and incubated on ice for 15 minutes.
  • the tube was spun at 14,000 rpm at 4° C. for 5 minutes to pellet the precipitated supercoiled plasmid DNA.
  • the supernatant was discarded and the DNA pellet was washed with ice cold 70% ethanol.
  • the DNA was pelleted again by centrifuging at 14,000 rpm for 5 minutues at 4° C. The supernatant was discarded and the pellet was dried then dissolved into 250 ⁇ l of 1 ⁇ TE to form the primary plasmid library.
  • PshAI methylase selection 1 ⁇ g of DNA from each library was digested in 50 ⁇ l 1 ⁇ NEBuffer 2 with 8 U, 4 U, 2 U and 1 U of the PshAI endonuclease prepared in step 2 above respectively at 30° C. for 2 hours. 50 ng (2.5 ⁇ l) of the PshAI digested DNA was transformed into E. coli ER2416 and plated on L-broth plates containing 100 ⁇ g/ml ampicillin. A total of 108 transformants from the 3 libraries were analyzed as follows: Plasmid from each colony was isolated by miniprep procedure and digested with PshAI endonuclease.
  • pPshAIM19, pPshAIM20, pPshAIM26 and pPshAIM33 all from the HinP1I library, were found to be fully protected from PshAI digestion. Further restriction analysis showed that pPshAIM19 and pPshAIM20 were identical, containing a 3 kb insert, and that pPshAIM26 and pPshAIM33 were identical and contained a 4.5 kb insert.
  • pPshAIM26 contained the same 3 kb of Plesiomonas shigelloides as pPshAIM19 plus an additional 1.5 kb of DNA located 3' to the methylase gene.
  • DNA sequencing of the 3 kb insert on pPshAIM19 was performed using the CircumventTM DNA sequencing kit (New England Biolabs, Inc.; Beverly, Mass.) according to the manufacturers instructions.
  • Various HinP1I and Sau3AI subclones of pPshAIM19 were made in vector pUC19 and synthetic oligonucleotide primers were synthesized to accomplish the sequencing.
  • Miniprep DNA preparations of pPshAIM19 were used as templates.
  • the six frame amino acid sequence translated from the DNA sequence was compared with the homologous region of various methylases and motifs I and IV of m 6 A ⁇ -type methylase were identified.
  • the DNA sequence of motif I was found to be 5'-GGACAGTTTTTCACTCCA-3' (SEQ ID NO:9) and 5'-TTAGATCCA GCATGCGGCTCAGGAGGCTTTCTT-3' (SEQ ID NO:10), which translates into the amino acid sequence: GQFFTP (SEQ ID NO:11) and LDPACGSGGFL (SEQ ID NO:12), where the amino acids in bold match the conserved or nearly conserved residues.
  • the DNA sequence of motif IV was found to be 5'-GATGCGATACTCACAAACCCTCCGTTT-3' (SEQ ID NO:13) which translates into the amino acid sequence: DAILTNPPF (SEQ ID NO:14).
  • a putative start site ATG was identified 8 nucleotides away from a promising ribosome binding sequence: 5'-GGAGAG-3' (SEQ ID NO:15).
  • a putative stop site was also identified.
  • the methylase gene so identified is 1176 bp long.
  • ORF1 A 697 bp open reading frame, designated ORF1, was observed 3' to the methylase gene, oriented in the same direction as the methylase gene and separated from it by 48 nucleotides.
  • This ORF1 was cloned into the expression vector pAII17 to express the gene to test if it was the PshAI endonuclease.
  • Two DNA oligomer primers were synthesized to amplify ORF1 from Plesiomonas shigelloides DNA such that it could be placed into the NdeI site of the expression vector pAlI17. Although multiple clones of this gene were examined, no PshAI activity was observed.
  • the cell pellet (15 g) was resuspended in 50 ml of buffer A (20 mM Tris-HCl, 1 mM Dithiothreitol (DTT), 0.1 mM EDTA, pH 7.5) containing 1 mg/ml lysozyme and incubated on ice for 1 hour. 200 ⁇ l of 25% Triton-X100 was added to a final concentration of 0.1%. The cell suspension was frozen at -70° C., then thawed on ice. The extract was centrifuged at 15,000 rpm for 15 minutes at 4° C.
  • buffer A (20 mM Tris-HCl, 1 mM Dithiothreitol (DTT), 0.1 mM EDTA, pH 7.5
  • Triton-X100 25% Triton-X100 was added to a final concentration of 0.1%.
  • the cell suspension was frozen at -70° C., then thawed on ice.
  • the extract was centrifuged
  • This heparin-sepharose pool was diluted with 2 volumes of buffer A and applied to a 3 ml heparin-TSK FPLC column (TosoHaas; Philadelphia, Pa.) equilibrated in buffer A containing 100 mM NaCl.
  • the column was washed with 10 ml of buffer A containing 100 mM NaCl followed by a 40 ml linear gradient of 0.1M NaCl to 0.6M NaCl in buffer A. 1 ml fractions were collected. Fractions were assayed for PshAI activity with ⁇ DNA. The peak of restriction enzyme activity eluted between 0.32 and 0.35M NaCl and 3 fractions were pooled.
  • Amino terminal PshAI protein sequence The approximately 35 kD protein band obtained was subjected to amino terminal protein sequencing on an Applied BioSystems Division, Perkin-Elmer Corporation (Foster City, Calif.) Model 407A gas phase protein sequencer (Brooks, et al., Nucleic Acids Research, 17:979-997 (1989), the disclosure of which is hereby incorporated by reference herein). The sequence of the first 28 residues obtained was the following: MSILDNEKQLXILNIINEGVTPAIIPEL (SEQ ID NO:16).
  • the membrane was cut 1 cm larger then the gel on all sides (10 cm ⁇ 8 cm) and was placed on the bottom side of the gel. This was placed on top of a two inch high stack of paper towels. A glass plate was placed on the top of the stack and a small weight was placed on the plate. The DNA transfer was allowed to proceed overnight.
  • the immobulin S membrane was thoroughly dried then the DNA was UV cross-linked to the membrane by exposure to 33.33 joules/cm 2 UV light for 30 minutes in a UV crosslinker, X Linker-254 (Automated BioSystems, Inc.; Essex, Mass. and Owl Scientific, Inc.; Woburn, Mass.).
  • pPshAIM19 was digested with 20 U of Sau3AI for 1 hour at 37° C. in 100 ⁇ l reaction volume.
  • a 441 bp Sau3AI fragment from the end of the clone 5' to the methylase gene (bp 2354 to 2795, FIG. 2) was gel purified and resuspended in 37 ⁇ l of nuclease free dH 2 O (New England Biolabs, Inc.; Beverly, Mass.). This DNA was denatured at 95° C. for 5 minutes and then biotinylated according to the manufacturer's instructions (NEBlotTM PhototopeTM Kit; New England Biolabs, Inc.; Beverly, Mass.).
  • the blot from section A was wet with 6 ⁇ SSC at room temperature for 2 minutes then prehybridized at 68° C. in 8 ml of prehybridization solution composed of 5 ⁇ Denhardt's solution, 6 ⁇ SSC, 0.5% SDS, and 100 ⁇ g/ml denatured herring sperm DNA for 1 hour.
  • the biotinylated probe was denatured at 95° C. for 5 minutes then mixed with 8 ml prehybridization solution to a final concentration of 20 ng/ml and the blot was transferred to this solution.
  • the hybridization was carried out at 55° C. overnight.
  • the blot was then washed with two changes of 250 ml 2 ⁇ SSC, 0.1% SDS for 5 minutes each at room temperature followed by two changes of 250 ml 0.2 ⁇ SSC, 0.1% SDS for 15 minutes each at 55° C.
  • AvrII digested DNA was circularized at a concentration of 1 ⁇ g/ml in 1 ⁇ ligase buffer using 4000 U of T4 DNA ligase at 16° C. overnight. Enzyme was removed by extracting once with equlibrated phenol:CHCl 3 (50:50, v/v) and once with CHCl 3 . The DNA was precipitated by adding 1/10th volume of 5M NaCl and 1 volume of 2-propanol, pelleted by centrifugation, washed with ice cold 70% ethanol and dried. It was then resuspended in 50 ⁇ l of 1 ⁇ TE buffer and used as the template in the following inverse PCR reaction.
  • the PCR amplification conditions were: 95° C. for 2 minutes for one cycle, followed by 25 cycles of 95° C. for 20 seconds, 56° C. for 30 seconds and 72° C. for 3 minutes. 10 ⁇ l of the PCR reaction was analyzed by electrophoresis on a 0.8% agarose gel. A prominent band of approximately 2.9 kb was observed, as was some primer dimer product. To obtain a greater quantity of this product, the 2.9 kb band was gel purified and used as the template in subsequent PCR reactions carried out as above. The 2.9 kb product obtained was gel purified and resuspended in 50 ⁇ l of 1 ⁇ TE.
  • the 2.9 kb PCR amplified product was mapped using various endonucleases to find convenient sites for cloning.
  • a DraI site was found 400 bp from the 5' end of pPshAIM19 and an EcoRi site was located 1400 bp from the 5' end of pPshAIM19 (FIG. 3).
  • 4 ⁇ g of the 2.9 kb amplified product was digested with EcoRI and DraI in 100 ⁇ l reaction volume at 37° C. for 1 hour. The reaction was extracted once with equlibrated phenol:CHCl 3 (50:50 v/v), and once with CHCl 3 .
  • DNA was precipitated with 1/10th volume of 5M NaCl and 1 volume of 2-propanol, pelleted by centrifugation and washed with ice cold 70% ethanol.
  • the DNA was resuspended in 40 ⁇ l of 1 ⁇ TE and 2 ⁇ l was ligated into pUC19 (50 ng previously digested with HincII and EcoRI and gel purified) using 400 U T4 DNA ligase in 20 ⁇ l volume at 16° C. for 2 hours. 10 ⁇ l of ligation mixture was transformed into ER 2426 and plated on L-broth plates containing 100 ug/ml ampicillin for individual colonies. The plasmids were isolated by performing minipreps. The desired construct was identified by restriction enzyme digestion.
  • DNA sequencing was performed using the CircumventTM DNA sequencing kit (New England Biolabs, Inc.; Beverly, Mass.) according to the manufacturer's instructions, using M13/pUC primers NEB#1224 and NEB#1233 as well as custom synthesized primers.
  • PCR reaction condition was as follows:
  • the PCR amplification conditions were: 95° C. for 2 minutes for one cycle, followed by 20 cycles of 95° C. for 30 seconds, 56° C. for 30 seconds and 72° C. for 1.6 minutes.
  • DNA sequencing was performed using CircumventTM DNA sequencing kit (New England Biolabs, Inc.; Beverly, Mass.) according to the manufacturers instructions. Various subclones of this 1.5 kb PCR product were made in vector pUC19 and sequenced using M13/pUC primers NEB# 1224 and NEB# 1233. Miniprep DNA of the sub clones was used as template.
  • PshAI Restriction-Modification system Two open reading frames were found 5' to the methylase gene by examining the 6 frame amino acid translation of the DNA sequence (FIG. 3). An open reading frame, ORF3 of 237 bp was located 152 bp 5' to the methylase gene and running in the opposite orientation. The amino acid sequence of this open reading frame shows strong homology to other putative restriction control, or C, proteins (Tao, et al., J. of Bacteriology, 183:1367-1375 (1991), the disclosure of which is hereby incorporated by reference herein), and was most homologous to the EcoRV C protein. FIG. 4 shows an alignment of the PshAI C protein with EcoRV C protein.
  • the second open reading frame, ORF2 was located 11 bp 3' to the C gene, was 888 bp in length and was oriented in the same direction as the C gene.
  • This orf was identified as the PshAI endonuclease gene since the amino acid sequence deduced from the DNA sequence at the 5' end of this open reading frame matched exactly with the first 28 residues from N-terminus amino acid sequencing of the PshAI endonuclease gene.
  • the ambiguous residue X at position 11 of the amino acid sequencing results was found to be an arginine.
  • pPshAIM19 DNA was digested with PstI and X baI and the resulting 1.6 kb fragment containing the entire methylase gene was gel purified. This 1.6 kb fragment was ligated to vector pSX34lacZ ⁇ previously cleaved with the same enzymes and gel purified. The ligation reaction was transformed into ER2426 and approximately 300 colonies were obtained. The colonies were washed off the agar plate with 3 ml of 10 mM Tris, 10 mM MgCl 2 , pH 7.5 and inoculated into 50 ml of L-broth containing 25 ⁇ g/ml chloramphenicol and grown at 37° C. overnight with shaking.
  • the cells were pelleted by centrifugtion at 5,000 rpm for 5 minutes and the plasmid DNA was purified by a maxi-miniprep procedure (5 ⁇ scale of the miniprep procedure described above). Approximately 1.5 ⁇ g of purified DNA was digested with 20 U of PshAI in 100 ⁇ l volume at 30° C. for 1 hour, then phenol/chloroform extracted and precipitated with 1/10th volume of 5M NaCl and 1 volume of 2-propanol. Approximately 600 ng of the PshAI digested DNA was transformed into ER2426 and plated on L-broth plates containing 25 ⁇ g/ml chloramphenicol for individual transformant.
  • the restriction endonuclease gene was expressed by inserting the gene into a expression vector, pRRS, directly downstream of a strong inducible promotor (PlacUV5) and strongly recognized ribosome binding site.
  • pRRS a strong inducible promotor
  • lacUV5 strongly recognized ribosome binding site
  • two oligonucleotide primers were made utilizing the DNA sequence data.
  • the first oligonucleotide primer contained a PstI site to facilitate cloning, a stop codon in frame with the lacZ gene to terminate translation of the lacZ protein, a strong recognized ribosome binding site, seven nucleotide spacer between the rbs and the ATG start codon of the PshAI gene, a change in codon usage for serine at residue 2 to an E. coli preferred codon and sequence complementary to Plesiomonas shigelloides DNA for hybridization:
  • the reverse primer was designed to hybridize approximately 200 bp 3' to the 3' end of the endonuclease gene and had BamHI and EcoRI sites added to facilitate cloning:
  • the amplification product of approximately 1.1 kb was gel purified, cleaved with PstI and BamHI, phenol-chloroform extracted, precipitated, resuspended in 1 ⁇ TE and ligated into pRRS vector previously cleaved with PstI and BamHI and gel purified. The ligation reaction was transformed into E.
  • the PshAI restriction endonuclease may be produced from NEB #984 by propagation to mid-log phase in a fermenter containing L-broth medium with ampicillin (100 ⁇ g/ml) and chloramphenicol (25 ⁇ g/ml). The culture is induced by the addition of IPTG to a final concentration of 0.3 mM and allowed to continue growing overnight (16 hours). The cells are harvested by centrifugation and may be stored at -20° C. or used immediately.
  • Purification of the PshAI restriction endonuclease from NEB#948 can be accomplished by a combination of standard protein purification techniques, such as affinity-chromatography or ion-exchange chromatography, as outlined in step 6 above.
  • the PshAI restriction endonuclease obtained from this purification is substanially pure and free of non-specific endonuclease and exonuclease contamination.

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2537925A1 (fr) 2005-08-04 2012-12-26 New England Biolabs, Inc. Nouvelles endonucléases de restriction, ADN codant ces endonucléases et procédés pour identifier de nouvelles endonucleases avec la spécificité identique ou variée
EP2540823A1 (fr) 2005-08-04 2013-01-02 New England Biolabs, Inc. Nouvelles endonucléases de restriction, ADN codant ces endonucléases et procédés pour identifier de nouvelles endonucleases avec la spécificité identique ou variée
EP2548953A1 (fr) 2005-08-04 2013-01-23 New England Biolabs, Inc. Nouvelles endonucléases de restriction, ADN codant ces endonucléases et procédés pour identifier de nouvelles endonucleases avec la spécificité identique ou variée
EP2562252A1 (fr) 2005-08-04 2013-02-27 New England Biolabs, Inc. Nouvelles endonucléases de restriction, ADN codant ces endonucléases et procédés pour identifier de nouvelles endonucleases avec la spécificité identique ou variée
EP2565266A1 (fr) 2005-08-04 2013-03-06 New England Biolabs, Inc. Nouvelles endonucléases de restriction, ADN codant ces endonucléases et procédés pour identifier de nouvelles endonucleases avec la spécificité identique ou variée
EP2565267A1 (fr) 2005-08-04 2013-03-06 New England Biolabs, Inc. Nouvelles endonucléases de restriction, ADN codant ces endonucléases et procédés pour identifier de nouvelles endonucleases avec la spécificité identique ou variée
EP2568040A1 (fr) 2005-08-04 2013-03-13 New England Biolabs, Inc. Nouvelles endonucléases de restriction, ADN codant ces endonucléases et procédés pour identifier de nouvelles endonucleases avec la spécificité identique ou variée
EP2574668A1 (fr) 2005-08-04 2013-04-03 New England Biolabs, Inc. Nouvelles endonucléases de restriction, ADN codant ces endonucléases et procédés pour identifier de nouvelles endonucleases avec la spécificité identique ou variée

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